Probiotic Bacteria and their Supernatants Protect Enterocyte Cell Lines from Enteroinvasive Escherichia coli (EIEC) Invasion

Probiotic microorganisms have attracted a growing interest for prevention and therapy of gastrointestinal disorders. Many probiotic strains have been shown to inhibit growth and metabolic activity of enteropathogenic bacteria as well as their adhesion and invasion to intestinal cells. In the present study, we evaluated the interference of bacteria-free supernatants (BFS) of cultures belonging to sixteen strains of lactobacilli and bifidobacteria, with invasion of enteroinvasive Escherichia coli (EIEC) strain, using human colonic adenocarcinoma cell lines, T84 and Caco2 cells. To assess invasion of Caco-2 and T84 cells by EIEC, and measure the number of pathogens inside the enterocytes, the gentamicin protection assay was conducted. In addition, three different invasion inhibition assays were designed; namely co-incubation, pre-incubation and treatment with the BFS of probiotics. Data obtained and theoretical calculation showed that the most effective assay in the prevention of pathogen invasion was treatment with BFS. Besides, co-incubation assay was more valid than pre-incubation assay in invasion prevention. The obtained results suggest that probiotics may produce some metabolites that strongly prevent invasion of enteroinvasive E.coli into the small and large intestine. Also, probiotics are able to compete with or exclude pathogen invasion.

shows the list and source of strains.

Probiotic culture condition
All probiotic strains were cultured in the Man- To ensure the cell free status of BFS, supernatants were passed through a 0.4 μm pore size filter.

Cell lines culture
The human colon adenocarcinoma cell line To conduct an assay, each cell line was seeded at a density of 2×10 5 cells in 6-well tissue culture plates. The culture was refreshed every 2 days to form a monolayer culture, and was further cultivated for 7-10 days to reach confluent and differentiated cells. Then, monolayer cultures were incubated in an antibiotic-free medium for 24 h prior to treatment of the cells with bacteria, and cell invasion assay (7).

Invasion assays
To evaluate invasion of Caco-2 and T84 cells by EIEC, and measure the number of pathogens inside the enterocytes, the gentamicin protection assay was conducted with some modifications.
Briefly, 3×10 7 cfu/ml pathogen was added to each well. After 3 h incubation period, monolayers were washed 3 times with phosphate-buffered saline (PBS) and new media plus 50 l of 1 mg/ml gentamicin (Sigma G1379) was added to each well to kill bacteria outside the enterocytes, but not those within the cells. Then, incubation was further continued for an hour. Afterwards, wells were washed 3 times with PBS and tissue culture cells were lysed by adding 2 ml 5% Na-deoxycholate (Sigma D 6750). Note that E. coli is resistant to sodium deoxycholate. Subsequently, a ten-fold dilutions of well contents were prepared and plated out on Mueller-Hinton agar. After overnight incubation at 37 o C in aerobic atmosphere, the number of colony forming unit was determined, and the total internalized pathogen was calculated (8).

EIEC invasion inhibition assay
Three different invasion inhibition assays were designed, namely co-incubation, preincubation, and treatment with the bacteria free supernatant of probiotics to investigate differentiation, competition, exclusion, and probiotic BFS effect on EIEC (10). Besides, the intestinal cells and pathogenic bacteria without probiotic treatment were used as control.
In the co-incubation assay, either Caco2 cells or T84 cells were cultured, and then washed as described previously. Subsequently, 3×10 7 cfu/ml of pathogen and 3×10 7 cfu/ml of probiotic bacteria were added simultaneously to each cell line, and incubated for 3 h under optimal conditions.
In the pre-incubation assay, probiotics were added to cell lines, and allowed to adhere to the cells for 3 h. Then, each well was washed with PBS and new medium plus 3×10 7 cfu/ml pathogen was added into the wells and afterwards, incubation was further continued for 3 h.
In the supernatant treatment assay, the pathogen was treated with BFS from overnight culture of probiotic bacteria for 1 h. After treatment, the viability testing of the pathogen was performed. Then, the treated pathogen was added to the cell line at 3×10 7 cfu/ml as previously described.
Eventually, the gentamicin protection assay was conducted and the number of internalized pathogens was determined by plating serial dilution on Muller Hinton agar. Each assay was performed 3 times in duplicate (9).

Data analysis
All data are expressed as means structural equation modeling (SEM). Statistical analysis was performed by repeated measures. Analysis of variance (ANOVA) and p-values < 0.05 were considered to be statistically significant.

Invasion assay using T84 cells
The cfu/ml number of internalized E. coli without any treatment of cell line or pathogen was 2×10 6 .
Co-incubation of probiotic strains was able to significantly reduce numbers of E. coli inside the T84 cells by at least two log cfu/ml. Among those tested, L. acidophilus showed less activity. Pre-  The above table shows the number of internalized E. coli colonies recovered at the end of each assay. The log 10 cfu/ml of internalized E. coli without any treatment of cell line or pathogen was 2 ×10 6 .
The most effective assay in prevention of pathogen invasion was treatment with BFS where all strains showed the same effect except Bifidobacteria. In addition, the most effective strains were L. plantarum 5C, B. longum T in coincubation and L. plantarum 5C, L. casei Shirota 3D in pre-incubation, respectively. Moreover, coincubation assay was more effective than preincubation assay in invasion prevention. In our previous studies, we have shown the antibacterial activity of these sixteen probiotic strains (10). However, anti-invasive activity of probiotics and their BFS seems to be a secondary effect due to their bactericidal activity (3).

Discussion
Another anti-invasion mechanism could be due to an anti-adhesive effect of probiotic bacteria or their BFS, through blocking of epithelial surface receptors by binding of probiotic strains and their metabolite to these receptors or by binding to the respective ligands of the invasive bacteria.
In the present study, treatment of the pathogen with BFS has been very effective to prevent invasion. It seems that the main mechanism for preventing pathogen invasion is not competitive inhibition of receptor adhesion, but through changes to the environment, cell barrier or gene expression by probiotic-induced metabolites. In conclusion, in our study, we have explained that mechanisms involved in the pathogenic microbial invasion of eukaryotic cells are different depending upon the types of host tissues and microbial determinants. In future studies, we aim to investigate the mechanism of the anti-invasion activity of the most effective probiotic on Caco2 and T84 cells using molecular methods.